SUMMARY Soluble Amyloid-β oligomers (Aβo) trigger Alzheimer’s disease (AD) pathophysiology and bind with high affinity to Cellular Prion Protein (PrPC). At the post-synaptic density (PSD), extracellular Aβo bound to lipid-anchored PrPC activates intracellular Fyn kinase to disrupt synapses. Here, we screened transmembrane PSD proteins heterologously for the ability to couple Aβo–PrPC with Fyn. Only co-expression of the metabotropic glutamate receptor, mGluR5, allowed PrPC-bound Aβo to activate Fyn. PrPC and mGluR5 interact physically, and cytoplasmic Fyn forms a complex with mGluR5. Aβo–PrPC generates mGluR5-mediated increases of intracellular calcium in Xenopus oocytes and in neurons, and the later is also driven by human AD brain extracts. In addition, signaling by Aβo–PrPC–mGluR5 complexes mediates eEF2 phosphorylation and dendritic spine loss. For mice expressing familial AD transgenes, mGluR5 antagonism reverses deficits in learning, memory and synapse density. Thus, Aβo–PrPC complexes at the neuronal surface activate mGluR5 to disrupt neuronal function.

Invadopodia are matrix-degrading membrane protrusions in invasive carcinoma cells. The mechanisms regulating invadopodium assembly and maturation are not understood. We have dissected the stages of invadopodium assembly and maturation and show that invadopodia use cortactin phosphorylation as a master switch during these processes. In particular, cortactin phosphorylation was found to regulate cofilin and Arp2/3 complex–dependent actin polymerization. Cortactin directly binds cofilin and inhibits its severing activity. Cortactin phosphorylation is required to release this inhibition so cofilin can sever actin filaments to create barbed ends at invadopodia to support Arp2/3-dependent actin polymerization. After barbed end formation, cortactin is dephosphorylated, which blocks cofilin severing activity thereby stabilizing invadopodia. These findings identify novel mechanisms for actin polymerization in the invadopodia of metastatic carcinoma cells and define four distinct stages of invadopodium assembly and maturation consisting of invadopodium precursor formation, actin polymerization, stabilization, and matrix degradation.

RNA polymerase II requires multiple general transcription factors to initiate site-specific transcription. These proteins can assemble in an ordered fashion onto promoter DNA in vitro, and such ordered assembly may occur in vivo (Fig. 1a). Some general transcription factors can interact with RNA polymerase II in the absence of DNA, however, suggesting that RNA polymerase II may also assemble into a multi-component complex containing a subset of initiation factors before binding to promoter DNA (Fig. 1b). Here we present evidence from the yeast Saccharomyces cerevisiae for such an RNA polymerase II holoenzyme, a multi-subunit complex containing roughly equimolar amounts of RNA polymerase II, a subset of general transcription factors, and SRB regulatory proteins. Transcription by this holoenzyme is stimulated by the activator protein GAL4-VP16, a feature not observed with purified RNA polymerase II and general transcription factors alone. We propose that the holoenzyme is a form of RNA polymerase II readily recruited to promoters in vivo.

Caveolae are flask-shaped non-clathrincoated invaginations of the plasma membrane. In addition to the demonstrated roles for caveolae in potocytosis and transcytosis, caveolae may regulate the transduction of signals from the plasma membrane. Transformation of NIH 3T3 cells by various oncogenes-leads to reductions in cellular levels of caveolin, a principal component of the protein coat of caveolae. The reduction in caveolin correlates very well with the size of colonies formed by these transformed cells when grown in soft agar. Electron microscopy reveals that caveolae are morphologically absent from these transformed cell lines. These observations suggest that functional alterations in caveolae may play a critical role in oncogenic transformation, perhaps by disrupting contact inhibition in transformed cells.Oncogenic transformation leads to many changes in cultured cells. These changes include the loss of a requirement for growth factors (1), alterations in cell-surface molecules and membrane fluidity (2-4), the loss of contact inhibition of cell division and motility (5, 6), and the loss of anchorage dependence for growth (7). Measurements of these parameters are used to evaluate the oncogenic potential of a gene or its mutant derivatives. Despite the utility of these assays in scoring oncogenic potential in vitro, the molecular mechanisms that regulate these changes are poorly understood. A dissection of the molecular mechanisms behind these changes should lead to enhanced understanding of tumor initiation and progression in vivo.Caveolae are 50-to 100-nm flask-shaped invaginations of the plasma membrane (8, 9). Caveolae have been implicated in potocytosis and transcytosis of small molecules and ions in endothelial cells and sorting of surface proteins in polarized epithelial cells (8,(10)(11)(12)(13) (18,19). Finally, purified caveolar membrane domains are enriched in signaling proteins: Src-like nonreceptor tyrosine kinases, heterotrimeric G proteins, the ras-related GTPase Rap 1A, mitogen-activated protein kinases, other serine/threonine kinases, SH2-SH3 adaptor proteins, and other proteins with roles in signal transduction (20)(21)(22).The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.Caveolae may regulate the proliferation of normal cells in vitro. We have recently observed that NIH 3T3 cell lines transformed by several different oncogenes express greatly reduced levels of caveolin. Furthermore, the reduction of caveolin in these cell lines correlates with the size of colonies they form upon growth in soft agar. Electron microscopy reveals that caveolae are missing from the transformed cells that express reduced levels of caveolin. The reduction in caveolin does not correlate with serum requirements for growth of these cell lines in culture. The loss of caveolin could contribute to a loss of contact inhibition of motility or mitosis in tr...

The RNA polymerase II holoenzyme consists of RNA polymerase II, a subset of general transcription factors, and regulatory proteins known as SRB proteins. The genes encoding SRB proteins were isolated as suppressors of mutations in the RNA polymerase II carboxy-terminal domain (CTD). The CTD and SRB proteins have been implicated in the response to transcriptional regulators. We report here the isolation of two new SRB genes, SRB10 and SRB11, which encode kinase- and cyclin-like proteins, respectively. Genetic and biochemical evidence indicates that the SRB10 and SRB11 proteins form a kinase-cyclin pair in the holoenzyme. The SRB10/11 kinase is essential for a normal transcriptional response to galactose induction in vivo. Holoenzymes lacking SRB10/11 kinase function are strikingly deficient in CTD phosphorylation. Although defects in the kinase substantially affect transcription in vivo, purified holoenzymes lacking SRB10/11 kinase function do not show defects in defined in vitro transcription systems, suggesting that the factors necessary to elicit the regulatory role of the SRB10/11 kinase are missing in these systems. These results indicate that the SRB10/11 kinase is involved in CTD phosphorylation and suggest that this modification has a role in the response to transcriptional regulators in vivo.